Beta A4 amyloid protein and its precursor in Alzheimer's disease.

Pharmac.Ther.Vol. 56, pp. 97-117, 1992 Printed in Great Britain.All rightsreserved

0163-7258/92 $15.00 © 1992PergamonPressLtd

Specialist Subject Editor: C. BELL

flA4 AMYLOID PROTEIN A N D ITS PRECURSOR IN ALZHEIMER'S DISEASE ASHLEY I. BUSH,* KONRAD BEYREUTHER~" a n d COLIN L. MASTERS*~ *Department of Pathology, The University of Melbourne, Parkville, 3052 and The Mental Health Research Institute of Victoria, Royal Park Hospital, Parkville, 3052, Australia tCenter for Molecular Biology, University of Heidelberg, Germany Abstract--The flA4 amyloid protein is now understood to play a pivotal role in the development of Alzheimer's disease. This protein is generated by the abnormal processing of the amyloid protein precursor, a large membrane glycoprotein. Insights into the mechanisms of this abnormal processing will give information relevant to the design of new therapeutic strategies for Alzheimer's disease. CONTENTS 1. Introduction 2. flA4 Amyloid Protein 3. Amyloid Protein Precursor 3.1. The amyloid protein precursor gene and its products 3.1.1. Regulation of the amyloid protein precursor gene 3.1.2. Mutations of the amyloid protein precursor gene 3.1.3. Animal and cellular models for flA4 amyloidogenesis 3.2. Proposed functions of the amyloid protein precursor. 3.2.1. Amyloid protein precursor binding to elements of extracellular matrix 3.3. Processing of amyloid protein precursor 3.4. Amyloid protein precursor messenger RNA in Alzheimer's disease 4. Circulating Forms of APP as Indicators of Central APP Metabolism 4.1. APP in platelets 4.2. APP in nonplatelet blood elements 4.3. Plasma APP as a marker for AD Acknowledgements References

97 98 100 100 102 103 103 103 104 104 106 106 106 107 108 109 109

1. I N T R O D U C T I O N

Alzheimer's disease (AD) is the major form of adult-onset dementia. Because there is a direct and exponential correlation between age and the disease prevalence, the incidence of AD is increasing rapidly with the growth of the elderly population, creating a major public health problem. At present, the disorder is neither curable nor preventable. Approximately 5-10% of individuals in their 60s, and more than 20% of individuals in their 80s, are symptomatic with this form of progressive dementia, but there must be a large subclinical prevalence of AD. flA4 amyloid deposition in the brain, so far the only pathological hallmark of AD, is found in 45% of individuals over 50 years of age (Davies et al., 1988). Most cases of AD appear sporadic; however, about 10% :[:Corresponding author. Abbreviations--ACA, amyloid congophilic anglopathy; AD, Alzheimer's disease; APCs, amyloid plaque cores; APP, amyloid protein precursor; DS, Down's syndrome; FAD, familial Alzheimer's disease; HCHWAD, hereditary cerebral hemorrhage with amyloidosis-Dutch type; HSPG, heparin sulfate proteoglycans; IgG, immunoglobulin G; KPI, kunitz protease inhibitor; L-APP, leukocyte APP; mRNA, messenger RNA; NFTs, neurofibrillary tangles; PHFs, paired helical filaments; PN-II, protease nexin II; SAP, serum amyloid P component; SEC, serpin inhibitor-enzyme complex. 97

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of cases suffer from familial AD (FAD) with an autosomal dominant inheritance pattern. AD also invariably occurs in Down's syndrome (DS) where pathological changes, accelerated by some thirty years, become evident as early as the second decade of life (Rumble et aL, 1989).

2. flA4 AMYLOID PROTEIN In recent years much effort has focused on the role of brain flA4 amyloidogenesis in AD. flA4 is a 39-43 amino acid protein of molecular weight ~ 4 kDa which polymerizes as the major component of amyloid plaque cores [APCs] (Masters et al., 1985b) and amyloid congophilic angiopathy [ACA] (Glenner and Wong, 1984). It is derived by aberrant proteolytic cleavage of a much larger molecule, the amyloid protein precursor (APP), which has the structural characteristics of an integral transmembrane receptor (Kang et al., 1987). flA4 is also a component of neurofibrillary tangles [NFTs] (Masters et al., 1985a; Hyman et aL, 1989; Caputo et al., 1992). APCs, ACA and NFTs are the predominant neuropathological lesions in AD and DS, and are also found in dementia pugilistica (Allsop et al., 1990). These abnormal deposits, with accompanying neuronal and synaptic loss, occur most frequently in the hippocampus and temporal cortex (Price et al., 1991), which may explain the dysmnesia that occurs in AD, although the correlation of plaque density with neuropsychological deficits is still contentious (Terry et al., 1991). The biochemical basis for flA4 deposition is central to the pathogenesis of AD for several reasons: the synthetic flA4 molecule is highly self-aggregating, forming insoluble polymers which are difficult to metabolise (Masters et al., 1985b; Castafio et al., 1986; Kirschner et al., 1987; Hilbich et aL, 1991); synthetic fragments of the flA4 sequence are reported to have neurotoxic properties in neuronal cell culture and when injected into the neuropil (Kowall et al., 1991; Yankner et al., 1989; Frautschy et al., 1991); in DS there is an increase in APP messenger RNA (mRNA) (Tar~zi et al., 1987b) and APP protein levels accompany an acceleration of age-related dementia and flA4 deposition (Rumble et aL, 1989). The most compelling evidence comes from the recent observations that some forms of autosomal dominant FAD and congophilic angiopathy are associated with mutations of the APP gene causing amino acid substitutions in regions of the molecule associated with the fl A4 domain. Conversely, such mutations have not yet been found in nonAD subjects (van Broeckhoven et al., 1990; Levy et al., 1990; Goate et al., 1991; Chartier-Harlin et al., 1991; Naruse et al., 1991; Murrell et al., 1991). Finally, flA4 amyloid deposition is currently the only specific pathological feature of AD (Joachim and Selkoe, 1992). flA4 deposition in the brain in AD is accompanied by neuronal and synaptic loss, as well as the accumulation of other proteins in the intracellular and extracellular compartments of the brain cortex. Neuronal loss is restricted to certain areas of cerebral cortex, and is more pronounced in the subtype of AD where clinical onset is before the age of 65. Areas that are more severely affected by neuronal loss include portions of the medial temporal lobe, including hippocampus and amygdala, as well as subcortical nuclei that project to the cortex including the basal forebrain, locus ceruleus and the median raphe nucleus (Joachim and Selkoe, 1992). Neuronal loss involving several neurotransmitter and neuropeptide systems have been described: cholinergic, monoaminergic, glutamatergic, GABAergic, somatostatin, neuropeptide Y, corticotropin releasing factor and substance P (reviewed in Joachim and Selkoe, 1992). NFTs are characteristic of AD, but, in contrast to flA4 collections, are not as specific. NFTs can be found in several other neurodegenerative disorders that are not usually characterized by extracellular flA4 deposition. In AD, NFTs occur predominantly in the cerebral cortex, hippocampus, amygdala, basal forebrain and brainstem. Ultrastructurally, NFTs are composed of pairs of 10 nm twisted filaments, the so-called paired helical filaments (PHFs). Whether NFTs contain or are associated with intraneuronal flA4 accumulation is still debated, but considerable evidence has accumulated to support the presence of flA4 within or bound to NFTs (Masters et al., 1985a; Guiroy et al., 1987; Hyman et al., 1989; Grundke-Iqbal et al., 1989; Yamaguchi et al., 1990; Ito et aL, 1991; Perry et al., 1992; Caputo et aL, 1992). PHFs have been shown to contain or be tightly bound to the carboxyl one-third of the microtubule-associated protein, tau (Kondo et al., 1988; Wischik et al., 1988), a region that contains the microtubule binding domain (Lee et al., 1989). The tau associated with PHFs is abnormally hyperphosphorylated, variably soluble, aggregated,

flA4 amyloid protein and Alzheimer's disease

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abnormally located in the cell body rather than the axon and has altered electrophoretic mobility (Grundke-Iqbal et al., 1986a, b; Lee et al., 1989). To date there is no satisfactory explanation for the coincident depositions of flA4 and abnormal tau in AD brain. Other proteins that are associated with NFTs include neurofilaments, ubiquitin, MAP 5, tropomyosin, synaptophysin, cholinesterases, serum amyloid P (SAP), heparan sulfate proteoglycan (HSPG) and basic fibroblast growth factor. flA4-containing plaques of several different morphologies have been described which may reflect the possibility that flA4 amyloid plaques take years, if not decades, to mature. Studies of the evolution of flA4-related pathology in the brains of individuals with DS have helped to elaborate the maturation sequence of the flA4 deposits. The earliest lesion appears to be the diffuse, nonfibrillar plaque which consists of focal infiltration of the flA4 deposits extracellularly, with few or no surrounding degenerating neurites (Davies et al., 1988; Tagliavini et al., 1988; Yamaguchi et al., 1988; Joachim et al., 1989b). This is the predominant plaque type in AD and causes little structural alteration in the neuropil, flA4 deposition has been shown to precede the synaptic loss, but the reverse has not been demonstrated. Diffuse plaques have been reported to appear long before mature neuritic plaques or NFTs (Giaccone et al., 1989; Mann et al., 1990; Mann and Esiri, 1989). With progression of the disease, 'neuritic' or 'primitive' plaques are more abundant. These flA4 deposits are surrounded by abnormally swollen, dystrophic neurites (both axons and dendrites), which can be immunocytochemically labeled by antibodies to tau, PHFs and ubiquitin. A single cortical plaque may contain degenerating neurites of several different neurotransmitter specificities (Struble et al., 1987). The flA4 in neuritic plaques is compacted into dense cores which contain 7-10 nm filaments containing flA4 in the fl-pleated sheet conformation, characteristic of all amyloid depositions. These plaques appear to cause substantial disruption to the neuropil and are often associated with surrounding reactive astrocytes and microglial cells. flA4 deposits in APCs and cerebral vessel walls are associated with various other proteins. These include ferritin, complement Clq and C3c, lysosomal proteases, protease nexin-1, 0q-ACT, cystatin C (y-trace), chromogranin A, epidermal growth factor receptor, cholinesterases, SAP, immunoglobulin G (IgG), HSPG and basic fibroblast growth factor. Also, the neurites surrounding both diffuse and mature plaques are associated with various fragments of APP (Palmert et al., 1988; Perry et al., 1988; Ishii et al., 1989; Ghiso et al., 1989; Shelton et al., 1990; Tagliavini et al., 1990, 1991; Takahashi et al., 1990a, b; Tate-Ostroff et al., 1990; Joachim et al., 1991; Arai et al., 1991; Cole et al., 1991; Cras et al., 1991; Masliah et al., 1991; Hyman et al., 1992; Kawai et al., 1992). The propensity for flA4 having a direct neurotoxic effect is still debated and is subject to further confirmation. Evidence to support a pathogenic role for flA4 comes mainly from the observation that it is toxic to mature neurons in culture. This effect has been localized to an I 1 amino acid internal sequence that is homologous to a conserved region in the tachykinin family of neuropeptides (Yankner et al., 1990). The flA4 peptide does not seem to interact directly with the NK1 tachykinin receptor that binds substance P. However, another recently characterized receptor for the serpin class of protease inhibitors binds both flA4 and tachykinins with high affinity (Joslin et al., 1991). This receptor, serpin inhibitor-enzyme complex (SEC), specifically recognizes an amino acid sequence that is conserved in serpin protease inhibitors, tachykinins, and flA4. This is the same sequence as that which is responsible for the neurotoxic effects of flA4. It is speculated that, in Alzheimer's disease, excessive generation of the flA4 peptide may competitively inhibit binding of the protease complex to SEC receptors and that self-aggregation of flA4 may permanently sequester SEC receptors, impairing the cellular capacity to clear extracellular proteases and allowing chronic proteolytic damage to occur which may lead to neuronal degeneration (Yankner and Mesulam, 1991). The cell of origin for the flA4 amyloidogenic substrate remains uncertain. There are four basic arguments in favor of a neuronal origin of the flA4 amyloid. First, there is a brain-restricted topography of the flA4 lesions which are deposited in a characteristic fashion: grey matter is much more affected than white matter; there is a laminar distribution of flA4 deposition within the cortical grey matter and a lack of spatial correlation between plaques and capillaries; there is an apparent spread of the lesions, beginning in the hippocampal/limbic areas, with a pattern of tangle-bearing neurons projecting into plaque-forming areas; vascular-associated deposits occur

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largely in the outer (adventitial) compartment rather than in the inner (intimal) layer; arteries and arterioles are more severely affected than capillaries and veins, with venules least affected by flA4 deposition; there is preferential deposition of flA4 in the Virchow-Robin space, with accentuation of vessels which penetrate the superficial areas of grey matter from the leptomeninges; vessels in the depth of sulci are more affected than vessels overlying gyri; there is a restriction of NFTs to cells of neuronal origin; the architecture of the extracellular space determines the morphology of the APCs. Overall, the spectrum of plaque morphologies suggests a process of para-amyloid infiltration before crystallisation into mature plaques. The second basic argument in favor of a neuronal origin of the flA4 amyloid is that there is a common flA4 subunit in the amyloid of intraneuronal tangles and extracellular plaques. If this subunit is derived from the same amyloid protein precursor (APP), then the source of APP is most likely to be neuronal. This argument is supported by the observations that the flA4 is derived from that part of the transmembrane domain of APP which has the topologic potential to partition into either the intracellular space (as NFTs) or into the extracellular space (as APCs or ACA) (Kang et al., 1987). Moreover, APP is expressed in neurons associated with the flA4 amyloid lesions (see above). The third basic argument in favor of a neuronal origin of the flA4 amyloid is the successful inducement of flA4 formation in the cerebral cortex of mice, transgenic for APP75j which is governed by a neural-specific promoter (Quon et al., 1991). The final argument in favor of a neuronal origin for flA4 amyloid is the observation that plaques and perivascular amyloid also occur in the PrP/scrapie model (Diedrich et al., 1991). There are also four basic arguments in favor of a hematogenous origin of the flA4 amyloid. First, amyloid congophilic angiopathies are p r i m a f a c i e evidence for a disturbance of flA4/APP metabolism within the vascular compartment, flA4 ACA occurs both as a sporadic disease and as a familial disease (hereditary cerebral hemorrhage with amyloidosis-Dutch type, HCHWA-D) (Luyendijk et al., 1988), and also occurs in association with cerebral vascular malformations (Hart et al., 1988). That amyloidosis can be manifested by vessel involvement is evidenced by such occurrences in other systemic amyloidoses such as AA amyloidosis (Prelli et al., 1987). The second basic argument in favor of a hematogenous origin of the flA4 amyloid is the fact that APP, with flA4 intact, exists in the circulation where it is found in platelets (Bush et al., 1990; Cole et al., 1990; Gardella et al., 1990; Schlossmacher et al., 1992). The presence of circulating full-length APP may account for the origin of the flA4 deposits which have been reported in skin and gastro-intestinal mucosa (Joachim et al., 1989a; Soininen et al., 1992). The third basic argument in favor of a hematogenous origin of the flA4 amyloid is the fact that the carboxyl terminus of ACA flA4 is attenuated compared to APC flA4. The ACA flA4 sequence ends after 39 or 40 residues (Joachim et al., 1988; Prelli et al., 1988), whereas the APC flA4 has 42 or 43 residues (Masters et al., 1985b). These observations support the possibility that perivascular flA4 amyloid may originate from a different source or by a different mechanism than the APC flA4 amyloid. The final basic argument in favor of a hematogenous origin of the flA4 amyloid is the fact that flA4 amyloid lesions are accompanied by several serum-derived adventitial molecules including ferritin, complement Clq and C3c, ~I-ACT, SAP, cystatin C (~-trace) and IgG.

3. AMYLOID PROTEIN PRECURSOR The sequence of flA4 was used to probe the human genome to reveal the full sequence of a larger protein, APP (Kang et al., 1987; Goldgaber et al., 1987; Tanzi et al., 1987b; Robakis et al., 1987b). The predicted structure of APP indicated that it is an integral transmembrane cell-surface receptor (Kang et al., 1987) but its function remains uncertain. The amyloidogenic flA4 region spans part (11-15 residues) of the transmembrane region and part (28 residues) of the adjacent extracellular domain of APP (Fig. 1). 3.1. THE AMYLOIDPROTEINPRECURSORGENE AND ITS PRODUCTS The APP gene is located on chromosome 21 at the boundary of 21q21.3 and 21 q22.1 (Kang et al., 1987; Tanzi et al., 1987b; Goldgaber et al., 1987; Robakis et al., 1987a; Zabel et al., 1987), very

flA4 amyloid protein and Alzheimer's disease Exon 16

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FIG. 1. Schematic diagram of the flA4 domain of APP (modified from Kang et al., 1987), showing the APP secretase site close to the exon 16-17 boundary; the sites of reported mutations of amino acids are shown encircled. The flA4 domain extends over 42/43 residues for the plaque cores (APC) and 39 residues for the congophilic angiopathy (ACA). close, if not within, the obligate DS region of the chromosome. The gene has 19 exons (Lemaire et aL, 1989) and at least six different mRNAs have been shown to be generated by alternate splicing (Figs 2 and 3). These are expressed as a complex family of 90-160 kDa membrane-bound and soluble glycoproteins. The prototype is APP695 (Kang et al., 1987). APP75t and APP770 are isoforms created by the insertion of a serine protease inhibitory domain of the Kunitz type II family (Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et aL, 1988). APPT,4 and APP770 contain an OX-2 related domain (Kitaguchi et al., 1988; Golde et al., 1990). The postulated splice product APP563 is homologous to APP75~ but is carboxyl-terminally truncated and therefore could only yield a nonamyloidogenic secreted form (DeSauvage and Octave, 1989). APP365 lacks both the Kunitz protease inhibitor (KPI) and flA4 domains (Jacobsen et aL, 1991). APP733(L-APP) mRNA is found in monocytes, activated T-cells and microglia. It resembles APP751 but lacks exon 15 (K6nig et aL, 1992). The region coding for the/~A4 amyloid extends over exons 16 and 17. The translation frame is such that flA4 could not be the product of an aberrant alternative splicing event. The APP gene is highly conserved in evolution. Mouse and rat APP69s have 22 and 18 amino acid substitutions, respectively, compared to the human APP695 (Yamada et al., 1987; Shivers et al., 1988). An APP homolog (APPL) has been described in Drosophila (Luo et al., 1990; Rosen et al., 1989), indicating Exon Structure

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FIG. 2. The overall exon structure of the APP gene is shown above the corresponding structural domains as currently elucidated. Numbers within boxes refer to the number of codons/amino acids. SP, signal peptide; CYS, cysteine rich domain; E/D, negatively charged (glu/asp) domain; KPI, Kunitz protease inhibitor domain; OX-2, neuro-immune homologous region; CHO, carbohydrate site; L-APP, leukocyte (T-cell and monocyte) domain which is removed by splicing in activated cells; TM, transmembrane domain; CD, cytoplasmic domain. JPT56/I--H

A. I. Busu et al.

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FIG. 3. The multiple isoforms of APP. The alternate splicing of the APP gene gives rise to at least nine forms of APP. The prototype, APP695 (Kang et al., 1987) is highlighted. The truncated forms (APP36s/563)are nonamyloidogenic. A recently described gene APLP (amyloid precursor-like protein), on chromosome 19, is shown for comparison. From Wasco et al. (1992, 1993). the ancient nature of this protein. In Drosophila APPL, the two regions with near 100% homology to the human APP sequence are in the intracytoplasmic domain, and in the negatively charged domain around residue 181, underlining the functional conservation of these regions of the protein. APP is found in human brain and cerebrospinal fluid (Weidemann et al., 1989), rat brain (Card et al., 1988) and human kidney, spleen, heart and adrenal tissues. Carboxyl terminus immunoreactivity is found in human serum where there is an elevation in DS (Rumble et al., 1989). APP751/770 have been shown to be identical with protease nexin-II (PN-II) which is secreted from human fibroblasts and forms inhibitory complexes with proteases including trypsin, chymotrypsin, the kallikreins, nerve growth factor- 7 subunit and epidermal growth factor binding protein (Van Nostrand et al., 1989; Oltersdorf et al., 1989). Northern blot analysis has detected APP mRNA in brain, kidney, heart, spleen, pancreas, muscle, thymus, lung adrenal gland, small intestine and liver (Tanzi et al., 1987b; Goldgaber et aL, 1987; Robakis et al., 1987a). The predominant forms of the protein product of these transcripts demonstrated to date are APP695, which is the main form in developing brain (Golde et al., 1990; Mita et aL, 1989; Kang and Miiller-Hill, 1990; Tanaka el al., 1988, 1989), and APP751/770which are the main forms in peripheral tissues (Golde et al., 1990; Mita et al., 1989; Neve et al., 1988; Tanaka et aL, 1988, 1989). In situ hybridization and immunocytochemistry have shown the neuron to be the main site of APP mRNA and protein production in the brain (Bahmanyar et al., 1987; Card et al., 1988; Goedert, 1987; Golde et al., 1990; Mita et al., 1989; Neve et al., 1988; Ohyagi et al., 1990; Schmechel et al., 1988; Shivers et al., 1988; Siman et al., 1989; Tate-Ostroff et al., 1989). Astrocytes and possibly oligodendrocytes contain APP mRNA and protein at lower levels than neurons (Mira et al., 1989). Astrocytes and microglia contain a higher proportion of the KPI-insert form of APP than neurons (Ohyagi et al., 1990), including L-APP (K6nig et al., 1992). 3.1.1. Regulation o f the A m y l o i d Protein Precursor Gene The APP gene promoter has the features of a housekeeping promoter, consistent with the ubiquitous expression of the APP gene. It lacks a typical TATA box and transcription initiates at multiple sites. The DNA sequence at the 5'-end identifies many elements which may be involved in transcriptional regulation of the APP gene (Salbaum et al., 1988). These include acute phase elements which may be involved in inflammatory conditions such as focal ischemia (Abe et al., 1991b), a heat shock element which may have a role in the induction of APP mRNA after heat stress in cultured human lymphoblastoid cells (Abe et al., 1991a), Hox 1.3 binding sites which may be involved in gene regulation during development, a transcription factor SP-I binding site and several GC-rich boxes which may be involved in tissue-specific transcription, a cyclic AMP


103

responsive element which may relate the gene to receptor signal transduction events, a CpG region which may facilitate gene regulation through methylation, and proto-oncogene related AP-1/FOS binding sites which may mediate the transcriptional induction of the APP gene by nerve growth factor or phorbol esters (Mobley et al., 1988). Translational regulation of APP may also play an important role. 3.1.2. Mutations o f the Amyloid Protein Precursor Gene

Linkage of FAD to chromosome 21 markers has been confirmed for some (Goate et al., 1989) but not all families with early-onset AD (Schellenberg et al., 1988). Late onset FAD has not yet shown linkage with chromosome 21 (Pericak-Vance et al., 1988; Schellenberg et al., 1988; St George-Hyslop et al., 1990). To date several mutations involving amino acid substitutions related to the fl A4 domain of APP have been linked to early onset FAD or HCHWA-D. The most common mutation is at codon 717 in the numbering sequence of APP770, resulting in substitutions of valine for isoleucine (Goate et al., 1991; Hardy et al., 1991; Naruse et al., 1991; Van Duijen et al., 1991), phenylalanine (Murrell et al., 1991) or glycine (Chartier-Harlin et al., 1991). This substitution occurs three residues from the carboxyl-terminus of the ~A4 sequence, within the transmembrane domain. Affected individuals are heterozygous for the point mutation and suffer early-onset dementia, but the pathological and clinical phenotypic expression of the disease is heterogeneous. The three codon 717 mutations have occurred in ethnically unrelated FAD kindreds. It has been proposed that codon 717 mutations could destabilize a regulatory stem-loop structure in APP mRNA and destroy an iron-responsive element (Tanzi and Hyman, 1991). It is speculated that this may dysregulate APP translation leading to an overproduction of APP as in DS. However, in situ hybridization on the brain of one patient with the APP717 Val to lie mutation has not indicated that the level of APP mRNA is altered (Harrison et al., 1991). HCHWA-D has been linked to a mutation at codon 693 of APP770 (Levy et al., 1990; van Broeckhoven et al., 1990). This leads to an amino acid substitution at position 22 within the flA4 domain. An early-onset FAD variant condition with pathological changes similar to HCHWA-D, but found in an unrelated family, has recently been linked to a mutation at codon 692 causing an Ala to Gly substitution (Hendriks et al., 1992). 3.1.3. Animal and Cellular Models for flA4 Amyloidogenesis The creation of a reliable model for/~A4 amyloidogenesis has proven to be a difficult task. An important consideration in judging the validity o f / / A 4 deposition in the brains of laboratory animals is to assess the contribution of incidental lesions such as corpora amylacea to any pathological findings in experimentally induced lesions. To date, only one laboratory has claimed successful /~A4 amyloid induction in transgenic animals. This was using transgenic mice with human APP751 under the control of a neural-specific promoter (Quon et al., 1991). These animals were 4-15 months in age. A cell culture model for amyloid fibril formation has been described using COS- 1 cells transiently transfected with cDNA encoding the carboxyl terminus 100 residues of APP (Maruyama et aL, 1990). A small proportion of the transfected cells developed perinuclear deposits that were immunoreactive with antibodies to the carboxyl-terminus of APP and to/~ A4. These results suggest that a single cleavage of APP at the amino terminus of/~A4 can generate a peptide potentially capable of forming amyloid fibrils. Aggregation of the carboxyl terminus 100 residues of APP is facilitated by oxidative stress (Dyrks et al., 1992), and in the future it may be possible to build on this observation to create an accurate in vitro model of the AD lesion. 3.2. PROPOSEDFUNCTIONSOF THE AMYLOIDPROTEINPRECURSOR

The predicted structure of APP695 indicates that it could serve as a cell-surface receptor (Kang et al., 1987), although its primary ligand has yet to be identified. APP has been described as being

highly expressed at points of neuronal contact in rat brain, suggesting a role in cell-cell contact

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(Shivers et al., 1988), an observation supported by studies of secreted APP which show that the protein promotes cell-cell and cell-surface adhesion (Schubert et al., 1989b; Breen et al., 1991; Chen and Yankner, 1991) and neurite outgrowth (Milward et al., 1992). The KPI-insert forms of APP have been reported to have a mitogenic effect (Schubert et al., 1989a). PN-II binds to proteases which may participate in neurite outgrowth including epidermal growth factor binding protein (Knauer and Cunningham, 1982) and nerve growth factor (Knauer et al., 1982), which may explain the growth enhancing effects of APP on fibroblasts (Saitoh et al., 1989). In addition, APP may inhibit a tissue kallikrein responsible for post-translational processing of the N G F precursor (Castro et al., 1990). These may be some of the means by which APP promotes neuritic branching in hippocampal cultures, enhance the survival of hippocampal neurons in vitro (Whitson et al., 1990, 1989) and mediate NGF-induced neurite outgrowth in cultured PCI2 cells (Milward et al., 1992). APP binds and inhibits a number of factors in plasma which participate in coagulation and wound repair, suggesting a modulating role for APP in these events. APP binds TGF-fl (Bodmer et al., 1990) which is released, along with other growth factors such as epidermal growth factor, by platelets following their activation. Kitaguchi et al. (1990) have demonstrated that the protease inhibitor region of APP inhibits coagulation factor Xa at an equilibrium dissociation constant of 1.2 x 10 -6 M. Similarly, a carboxyl terminus truncated 120 kDa form of APP released by HepG2 cells behaves as an inhibitor of coagulation factor XIa (Smith et al., 1990). In these latter two studies, the authors demonstrated that the proteolytic activity of thrombin is not inhibited by these APP forms. APP also inhibits the fibrinolytic enzyme, plasmin (Kido et al., 1990). APP is found in the extracellular matrix, where it is proposed to act as a signal for membrane extension (Klier et al., 1990). It is conceivable that the involvement of APP in modelling neuritic extension, synapse formation or regulation explains the effect of APP upon learning and memory models. Intraventricular infusions of antibodies to APP have been shown to impair the acquisition of a passive avoidance response in the rat (Doyle et al., 1990). Conversely, flA4 synthetic peptides have been claimed to induce amnestic effects in mice (Flood et al., 1991). L-APP, which lacks the 18 residues encoded by exon 15, has a varied expression between adherent and nonadherent T-lymphocytes (K6nig et al., 1992). This indicates that the function of exon 15 may concern cell contact or adhesiveness.

3.2.1. A m y l o i d Protein Precursor Binding to Elements o f Extracellular M a t r i x APP binds heparin (Schubert et al., 1989c), is associated with the extracellular matrix (Klier et al., 1990) and is involved in high affinity interactions with the basement membrane form of heparan sulfate proteoglycan (Narindrasorasak et al., 1991). If released at synaptic sites, the KPI-insert forms could participate in synaptic remodelling where serine proteases may play a critical role. It is interesting to observe that a protease suspected of releasing APP from the cell membrane may also act to release APP from the extracellular matrix (Small et al., 1992). Some authors have observed that highly sulphated glycosaminoglycans are found within the amyloid deposit of all forms of amyloidosis. They suggest that glycosaminoglycans are not only deposited coincidentally with the amyloid monomer, but may also play a significant role in altering the conformation of the molecule or its precursor (Kisilevsky and Snow, 1988). This may, in turn, promote the polymerization of the amyloid subunit. Heparan sulfate proteoglycans have been found to be associated with APCs and ACA even in primitive or diffuse collections and have been considered to be involved in early plaque formation (Snow et al., 1988). These authors propose that HSPGs may serve as atrophic factor in attracting neurites to the area of the plaque, thereby serving as a nidus for further plaque development to occur. HSPGs have been shown to promote neurite outgrowth in vitro (Lander et al., 1982).

3.3. PROCESSING OF AMYLOID PROTEIN PRECURSOR APP undergoes considerable post-translational modifications including tyrosine sulfation, Oand N-linked glycosylation (Oltersdorf et al., 1990; Weidemann et al., 1989), phosphorylation and


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sialic acid addition (Oltersdorf et al., 1990). The candidate kinases for phosphorylation include protein kinase C and calcium/calmodulin-dependent protein kinase II (Gandy et al., 1988). Once produced, the intact holoprotein is believed to be handled by more than one cellular processing pathway. In the neuron, the APP is anterogradely transported to synaptic endings where it colocalizes with synaptophysin (Koo et al., 1990) in vesicles (Schubert et al., 1991). APP may be stored in a soluble pool in these vesicles, as is the case with platelet a-granules, or it may be released from a membrane-bound fraction. This requires the action o f a postulated secretase which cleaves APP near the external surface of the membrane, leaving behind a fragment with the transmembrane domain and the carboxyl terminus. Evidence exists for the existence of an unidentified APP 'secretase' which cleaves APP at residue 16 of the//A4 domain, leaving a 10-17 kDa carboxyl terminal membrane-associated remnant (Sisodia et al., 1990; Esch et al., 1990). Recently, emphasis has been placed on the role of the endosomal/lysosomal system in the processing of APP into potentially amyloidogenic fragments. The cytoplasmic tail of APP contains a consensus sequence [NPXY] which signals rapid endocytosis. The lysosomal degradative pathway may then degrade the APP, yielding fragments which possess an intact/~A4 domain (Cole et al., 1989; Golde et al., 1992; Haass et al., 1992). Treating cells with leupeptin or ammonium chloride, agents which can partially inhibit lysosomal proteolysis, appears to enhance the amounts and stability of some of the carboxyl-terminus fragments (Golde et al., 1992). Carboxyl-terminus-containing fragments of APP that are larger than the nonamyloidogenic 'secretase' product, and hence potentially amyloidogenic, have been identified in human cerebral cortex (Nordstedt et al., 1991; Estus et al., 1992), purified cerebral microvessels (Tamaoka et al., 1992), and APP-transfected cell lines (Golde et al., 1992). Although several candidate proteases for the processing of APP have been proposed including acetylcholinesterase-associated protease (Small et al., 1991), Ca2+-dependent protease (Abraham et al., 1991), prolyl endopeptidase (Ishiiura et al., 1990), ingensin (Ishiura et al., 1989), calpain (Siman et al., 1990) and cathepsin B (Tagawa et al., 1991), an association between an APP-cleaving protease activity and clinical AD has yet to be shown. At least four mechanisms could lead to the generation of flA4. One possibility is that aberrant cleavage of APP may be caused by a defect in the normal constitutive proteolytic pathway that renders amyloid formation impossible by cleaving APP within the flA4 domain (Sisodia et al., 1990; Eschet al., 1990). If there is a failure of this proteolytic mechanism, then the APP may become a substrate for an alternative, flA4-generating, pathway. Conversely, there may be a modification to the APP molecule itself which then results in an alteration of the constitutive proteolysis. This mechanism is possible in the closely related disorder of familial Dutch congophilic angiopathy, which is associated with a mutation within the flA4 domain of APP (van Broeckhoven et al., 1990; Levy et al., 1990), and in forms of FAD where there are mutations close to the carboxyl terminus of the flA4 peptide (Goate et al., 1991; Chartier-Harlin et al., 1991; Naruse et al., 1991; Murrell et al., 1991; Lucotte et al., 1991). The processing of APP into various potentially amyloidogenic fragments may also be influenced by phosphorylation of the protein (Buxbaum et al., 1990). A third mechanism could involve an increase in APP substrate overwhelming the constitutive pathway and providing excess APP for an amyloidogenic pathway. This mechanism is likely in DS where an extra copy of chromosome 21 is associated with an increased expression of APP and the premature deposition of flA4 (Rumble et al., 1989). No such duplication of chromosome 21 has been observed in sporadic AD cases (Tanzi et al., 1987a; St George-Hyslop et al., 1987; Podlisny et al,, 1987), nor is there yet any convincing data to show an over-expression of all forms of APP in AD. A fourth possible mechanism is that constitutive processing of APP could yield soluble flA4 fragments or fragments which contain the flA4 domain. Soluble flA4 may aggregate into insoluble complexes as a product of elevated concentration, or they may become more self-aggregating and polymerize as a consequence of oxidative stress (Dyrks et al., 1992). The mechanisms by which the integral transmembrane forms of APP could be cleaved within the transmembrane domain to yield the carboxyl terminus of the flA4 monomer remains to be elucidated.

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Several lines of evidence support the possibility that flA4 amyioidogenesis is associated with a selective over-production of KPI-containing APP or an increase in the ratio of KPI-containing APP to APP695. Observations of a relative increase in 130 kDa KPI-containing plasma APP in AD (Bush et al., 1992) as well as the 2- to 3-fold increase in the levels of 133 kDa APP holoprotein in AD and aged brain (Nordstedt et al., 1991), are consistent with an increased production of fully-processed KPI-containing 130 kDa APP. KPI-containing forms of APP are present in the dystrophic neurites of amyloid plaques (Hyman et al., 1992). Although there is not yet a clear consensus, reports are accumulating that the proportion of KPI-containing APP mRNA relative to nonKPIcontaining APP mRNA is increased in sporadic AD (Tanzi et al., 1988; Tanaka et al., 1988, 1989; Johnson et al., 1988, 1990, 1989; Neve et al., 1988, 1990). A strong linear relation between increased APP751/APP695 mRNA ratio and increased plaque density in hippocampus and entorhinal cortex has been reported (Johnson et al., 1990). Increased mRNA and expression of KPI-containing APP released by lymphoblastoid cells from FAD cases has also been shown to be accompanied by aberrant intra-flA4 proteolysis (Matsumoto and Fujiwara, 1991). Finally, it has been shown that transgenic mice over-expressing KPI-containing APP75~ in the brain develop flA4 immunoreactive deposits (Quon et al., 1991) which are reminiscent of the early lesions seen in DS. 4. CIRCULATING FORMS OF APP AS INDICATORS OF CENTRAL APP METABOLISM The finding of APP in the blood carries important implications for the research on AD. Its presence yields an accessible source of APP to sample from living donors, its abundance and localization indicate that it has an important physiological function and the presence of fulllength APP in platelets means that a hematogenous source for the substrate of flA4 deposition is possible. 4.1. APP IN PLATELETS Carboxyl terminus-attenuated APP has been confirmed to be the predominant form of platelet APP and is released during platelet activation and degranulation (Bush et al., 1990; Van Nostrand et al., 1990; Smith et al., 1990; Cole et al., 1990; Gardella et al., 1990; Schlossmacher et al., 1992). This soluble species has been identified as PN-II (Van Nostrand et al., 1990) and as a coagulation factor XIa inhibitor (Smith et al., 1990), hence the APP species must be APP75t or APP770. mRNAs for APP695, APP751 and APP770 have been isolated from platelets (Gardella et al., 1990; Schlossmacher et al., 1992). The existence of full-length APP associated with platelet membranes has been confirmed (Cole et al., 1990; Gardella et al., 1990; Schlossmacher et al., 1992), as well as evidence that this species is shed during platelet activation (Cole et al., 1990; Gardella et al., 1990), although some observations oppose the likelihood of APP-containing microvesicle formation (Schlossmacher et al., 1992). There has been no report identifying a detectable quantitative or qualitative abnormality in platelet APP in AD. The function of APP in the platelet remains to be elaborated. The concentration of APP in the platelet indicates that it must have an important physiological role in events associated with coagulation. A breach of the vascular endothelial wall sets off a cascade of events which include the activation and aggregation of circulating platelets and the release of the glycoprotein contents of their 0t-granules. The proteins released by the platelets include substrates for the coagulation cascade and constituents of plasma that may be endocytosed into the e-granule (such as von Willebrand factor, fibrinogen, fibronectin, thrombospondin, factor V, high molecular weight kininogen, fl-thromboglobulin, prothrombin, platelet factor 4, albumin and IgG), and protease inhibitors (e-2-macroglobulin, e-l-antitrypsin, Cl-inhibitor and 0t-2-antiplasmin), as well as numerous growth factors (such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and transforming growth factor-il L the dual roles for which are the local repair of the defect and the promotion of reparative tissue proliferation. Although we were unable to detect significant differences between AD and control platelet forms of APP on immunoblotting (Bush et al., 1990), technical refinements may allow detection of


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differences between isoforms which correlate with the development or progression of the disease. Furthermore, the demonstration of a possible vesicular release of platelet APP with the carboxylterminus intact raises the likelihood that the circulating form of APP is the substrate for the proteolytic events that result in the release of flA4. flA4 deposits in skin, subcutaneous tissue, intestine and around extracranial blood vessels have been reported in tissue obtained from individuals with AD as well as elderly controls (Joachim et al., 1989a; Soininen et al., 1992). These authors, like others (Peterson et aL, 1986; Ueda et al., 1989), suggest that AD may be a systemic disease with peripheral manifestations. If confirmed, a likely source for these deposits would be APP derived from platelets. The variation in the fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene in labelled platelet membranes, an index of membrane fluidity, has been shown to be a stable familial trait that is associated with a clinically distinct aggressive subtype of AD (Zubenko et al., 1987b). A single genetic platelet membrane fluidity locus has been postulated, which appears to explain 80% of the total variation of membrane fluidity within the families of patients with AD (Chakravarti et al., 1989). The increase in platelet membrane fluidity associated with AD may result from an accumulation of an internal membrane compartment resembling smooth endoplasmic reticulum (Zubenko et al., 1987a; Hajimohammadreza et al., 1990). One report has demonstrated increased intracellular membrane fluidity in AD platelets but no increase in plasma membrane fluidity in AD platelets compared to controls (Piletz et al., 1991). A more recent report surveying platelets obtained from 95 AD subjects and 133 controls failed to discriminate the two clinical groups, although these authors did not discriminate between plasma membrane and internal membrane fluidity (Kukull et al., 1992). Platelet proteins may be synthesized in the megakaryocyte or synthesized de novo in the circulating platelet, which is known to contain mRNA, rough endoplasmic reticulum and polyribosomes (Kieffer et al., 1987). The possibility that a platelet membrane abnormality affects APP release and influences the consequent production of an amyloidogenic fragment warrants further investigation. The overexpression of APP in DS may be linked to the invariably premature development of AD lesions (Neve et al., 1988; Rumble et al., 1989; Tanzi et al., 1987b). The association of megakaryoblastic leukemia with DS (Lewis et al., 1983) could now be re-evaluated in the light of the present findings of the platelet as a major source of APP. 4.2. APP IN NONPLATELETBLOODELEMENTS The finding of low levels of APP in a proportion of lymphocytes has been substantiated elsewhere with the elucidation of a secreted and soluble form of APP in T-lymphocytes (Mrnning et al., 1990; Krnig et al., 1992). There is a very large increase in APP secretion from T-lymphocytes upon activation, but only low levels in the resting state. The question of the origin and nature of plasma APP immunoreactivity has been the focus of recent enquiry (Rumble et al., 1989). APP mRNA has been found in many extraneural tissues (Tanzi et al., 1987b, 1988) and, if transcribed, the resultant protein would more likely escape from tissues not constrained by the highly impermeable tight junctions of the blood-brain barrier. Endothelial cells constitutively express APP mRNA and these levels can be increased by treatment with interleukin-1 (Goldgaber et al., 1989). Human plasma contains forms of APP of similar size to those released by the platelet (Bush et al., 1992). These data, together with the observation of strong immunocytochemical labelling of megakaryocytes for APP, indicate that the platelet may be the major source of circulating APP, perhaps released by platelet destruction in the spleen. Studies to determine if the platelet is the origin of plasma APP involve assaying for plasma APP in various hematological conditions where there are platelet abnormalities. Preliminary data indicate that plasma APP species are decreased in thrombocytopenia, and increased in thrombocytosis (A. I. Bush, S. Whyte, K. Beyreuther and C. L. Masters, unpublished observations). Plasma APP is decreased in grey platelet syndrome, a disorder of platelet 0t-granule storage, where APP is not stored in the soluble fraction of platelets (Q. x. Li, A. I. Bush, K. Beyreuther and C. L. Masters, manuscript in preparation). Together, these data support a platelet origin for plasma APP, although the platelet may not be the sole contributor to plasma APP. Because identical molecular

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size species exist in brain, a contribution of these extravascularly derived soluble APP species to plasma APP is still possible. Although the APP species in plasma lack the intact/3 A4 domain, these forms are still of interest in AD research. The finding of APP in human and nonhuman plasma provides the researcher with access to the secretable products of APP processing in living subjects. The abnormal secretable products of aberrant APP cleavage may be evident in plasma in AD, and this provides a search strategy for a diagnostic marker for the disorder. 4.3. PLASMAAPP AS A MARKERFOR AD We have recently shown that there is a relative increase in the 130 kDa form and a decrease in the 65 and 42 kDa forms of APP in the plasma of moderately to severely demented AD patients (Bush et al., 1992). These observations may form the basis for a peripheral biochemical marker for AD. Prospective studies are required to determine the predictive value of the plasma APP profile for clinical or pathological outcome, but these may take many years to complete. It is anticipated that the proportion of 130 kDa or 42 kDa APP in plasma may predict individuals who are likely to develop/3A4 amyloid deposition, and we speculate that the controls who possessed proportions of APP beyond the recommended thresholds (10 of 77 controls for 130 kDa APP; 15 of 77 controls for 42 kDa APP) will be far more likely to develop AD pathology. As this assay is attempting to monitor the biochemical lesion underlying flA4 amyloidogenesis, it is not expected that the test could accurately predict which individual will dement or predict when dementia will occur, because the quantity of 13A4 deposition does not sensitively correlate with neuropsychological deficit (Terry et al., 1991) and there is a large variation between individuals as to when the lesions in the brain are severe enough to manifest themselves as a clinical deficit. It is certaintly possible to have abundant/3A4 deposition in the brain without evidence of cognitive decline (Davies et al., 1988). Perhaps a more reasonable expectation of this assay as a predictive test is that a threshold may exist which describes individuals who will never develop/3A4 amyloid or develop it later, rather than sooner, in life and hence are far less likely to suffer primary dementia. It is notable that using the recommended thresholds for the proportions of 130 and 42 kDa plasma APP described, a test is generated which has a false positive rate of 13-20% and a false negative rate of 20-26%. The false positives, individuals who have been diagnosed by the test as having AD but who are in fact clinically normal, may represent subclinical cases of AD. There is a large subclinical population with brain flA4 deposition (Davies et al., 1988) which must contribute to the false positive rate for this assay. In fact, more false positives would have been expected from the age-matched group. The observation that the spread of their results is similar to that of the younger adult control group probably reflects the stringent inclusion criterion for the age-matched control group, which would have excluded individuals with very mild dementia. The false negatives, individuals diagnosed as having clinical AD not in agreement with the test result, may reflect either the 10-15% of cases who are incorrectly diagnosed as having AD even on stringent clinical grounds or may also reflect subtypes of AD caused by variant biochemical lesions, for example mutations of the APP gene, which may not manifest themselves in the same way in plasma. These data are at variance with those of Podlisny et al. (1990) who reported no qualitative or quantitative differences in a soluble APP band of similar molecular weight (125 kDa) in AD plasma. This may be explained by the procedural differences between the studies. Podlisny et al. (1990) used plasma samples pooled from several individuals, which may have suppressed differences because there exists a considerable variance in the proportion of plasma APP species between cases. This, combined with the false positive and false negative problems described above, would have disadvantaged the ability of their assay to discriminate between clinical groups. There is substantially more APP in serum than in plasma, presumably released by platelet degranulation during clotting. The origin of plasma APP is unknown, but reflects a steady state level of APP expression that is lost when platelets release their contents into it. Platelet activation and degranulation is an event subject to large variance because platelet metabolism is readily inhibited, often for several days, by numerous environmental influences manifested in plasma, including common medicaments (for example, aspirin and nonsteroidal anti-inflammatory drugs),


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dietary factors (for example, garlic and alcohol), stress and tobacco (Siess, 1989). Therefore, serum, the fluid fraction of clotted blood, is not a good vehicle for determining the status of systemic APP secretion, although it is an easily-derived tissue sample which can provide a rich source of APP for physico-chemical studies. The ability of carboxyl-terminal and amino-terminal radioimmunoassays to differentiate APP levels in AD (Rumble, 1991) may have suffered from using serum as the biological sample instead of plasma, because it may have been measuring an immunoreactive component derived from variable platelet release rather than a baseline APP level. Several reports have now documented changes in cerebrospinal fluid levels of APP in AD, but a consensus has yet to emerge on the direction of these changes (Palmert et al., 1990; Prior et al., 1991). Palmert et al. (1990) describe alterations in the relative amounts of APP bands of 125, 105 and 25 kDa which could also be attributed to altered processing of APP. A recent report has described an elevation of a 133 kDa APP species from homogenized brain tissue of AD cases and elderly controls as compared to young controls, as assayed by western blot using an antiserum raised against the carboxyl terminus of APP (Nordstedt et al., 1991). These authors also report that there is no change in AD in the levels of an APP doublet migrating at 113 and 106 kDa. The increased ratio of 133 kDa to 113 and 106 kDa APP described in their report resembles the changes in the relative proportions of plasma APP species described by us (Bush et aL, 1992), with the exception that they were unable to discriminate AD cases from age-matched controls. Our estimates (Bush et al., 1992) of the concentration of 130 and l l 0 k D a plasma APP (approximately 50 pM) agree with previous estimates of plasma KPI-containing APP of similar molecular weight (Van Nostrand et al., 1991) suggesting, as others have (Podlisny et al., 1990), that KPI-containing APP may be the predominant form in plasma. Confirmation of this possibility by Western blot with a KPI-containing APP specific antibody has been achieved (Q.-X. Li, A. I. Bush, K. Beyreuther and C. L. Masters, manuscript in preparation). Therefore our observations are consistent with an increased production of fully-processed 130 kDa KPI-containing APP in AD. As outlined above, several lines of evidence support the possibility that flA4 amyloidogenesis is associated with an overproduction of KPI-containing APP or an increase in the ratio of KPI-containing APP to APP695. Acknowledgements--This work is supported by grants from the National Health and Medical Research

Council of Australia, the Aluminium Development Council of Australia and the Victorian Health Promotion Foundation. Konrad Beyreuther is supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium ffir Forschung und Technologie.

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A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation.

A4 protein precursor is increased by protein phosphorylation.

A4 region of the rabbit amyloid precursor protein gene.

A4 amyloid precursor protein.

Synthesis and secretion of Alzheimer amyloid beta A4 precursor protein by stimulated human peripheral blood leucocytes.

Amyloid precursor protein (APP) and beta A4 amyloid in the etiology of Alzheimer's disease: precursor-product relationships in the derangement of neuronal function.

Quantitative changes in the amyloid beta A4 precursor protein in Alzheimer cerebrospinal fluid.

Localization of Alzheimer beta A4 amyloid precursor protein at central and peripheral synaptic sites.

A4 protein precursor in the gerbil brain following transient cerebral ischemia.

A4 protein precursor is bound to neurofibrillary tangles in Alzheimer-type dementia.

A4-amyloid precursor protein: evidence for putative amyloidogenic fragment.

A4 amyloid precursor protein.

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

beta A4 amyloid protein deposition in brain after head trauma.

Stimulated platelets release amyloid beta-protein precursor.

Secretion of the beta-amyloid precursor protein.

A4 amyloid precursor protein in human brain: aging-associated increases in holoprotein and in a proteolytic fragment.

A4 protein precursor in the cytoplasm of neurons and senile plaque-associated astrocytes.

A4 protein precursor is widely distributed in both the central and peripheral nervous systems of the mouse.

A4 amyloid protein precursor.

Alzheimer amyloid beta-protein precursor in sperm development.

Beta-Amyloid Precursor Protein (βAPP) Processing in Alzheimer's Disease (AD) and Age-Related Macular Degeneration (AMD).

Abnormal and deficient processing of beta-amyloid precursor protein in familial Alzheimer's disease lymphoblastoid cells.

N-linked glycosylation of beta-amyloid precursor protein.